solid phase associated antibodies/ Jka antibodies

[elvallot]

Dear Forum Members

Hello, My name is Elizabeth Vallot and I work at North Oaks Medical Center in Hammond, LA. We currently use Capture and have for about a year now. I was wondering if your members have experieced an antibody dectected by solid phase but not present in tube in LISS or PEG. How are you handling these antibodies?

Also a few months back we had an antibody that matched Jka, when select cells that were Jka + were tested to confirm and Jka- cells were run to rule out other antibodies. Only the Jka + cells reacted. Upon testing the patient for Jka, she was Jka+. We had a cluster of these. The capture wells were hazy and showed a decreased cell button as compared to neg well control. Other patients had been recently transfused and unable to determine Jka status. When the patient in question was retested again (she was OB patient) about a week later her screen was neg. Any guesses as to what we are seeing?

[Yanxia]

can you exclude auto-antibody?

[BSIPHERD]

Was the DAT positive? Did you do an eluate? Had the patient received any IV IgG med? Had the patient received any blood products recently?

[jbrun]

We have been Capture users since the first of the year and have similiar experiences as you have described. We are struggling as to how to handle those patients with positive Capture results and negative tube testing. Autoantibodies were ruled out as well.

[Ann Viernes]

We have been using Capture R for about 15 years, and Galileo since 2005. Capture is known to pick up anti-Jka and anti-E not found by other methods. I can't explain a Jka pattern in a Jka+ person, however, other than a transient auto-Jka or an HLA antibody mimicking the Jka pattern on your panel.

As far as handling the solid phase positives that don't repeat in the tube, our policy is that when a solid phase antibody screen is positive in a person with no history of antibodies, we follow up with a LISS tube screen and a solid phase panel. If both are negative, the result is "SP+TTneg" or "Solid Phase Pos, Test Tube neg" interpreted as false positive and reported as negative.

We noticed that some of the patients with this result later developed an anti-E so we had a student do a master's project for her MS degree. She used 74 consecutive SP+TTneg specimens and ran PEG screens and panels. Of these, 11 were pos in PEG. Of the 11 pos, one was identified in PEG as anti-E, one as Anti-S, 2 with HLA ab, 3 QNS for further workup, 2 with no pattern, and 2 as false pos.

From June 2006 to July 2007, the SP+TTneg rate was 1%.

We are not routinely using PEG and will not as a result of the study, due to the increased sensitivity and false positives.

[yolis76]

I personally DO NOT LIKE solid phase for the very reasons that you listed. We encounter all of those issues, and it can feel like we are chasing our tails trying to find something that's not there.

About your issues with antibodies that are only reacting in SP, that's to be expected. You are using a more sensitive method and its possible that antibodies that are not detecable in the tube will be picked up in SP. All you can do is honor the antibody and give Ag neg units. I would not recommend doing XMs in SP because then you'll be looking for trouble.

About those situations in which you're sure you've identified an antibody and then the patient is ag+, its possible you've found a rare occurrence of an autoantibody with specificity other than Rh. The fact that the screen was neg later doesn't mean anything - once you've worked with SP some more you'll realize that the results are not reproducible, even on the same day with the same reagent and sample. It's frustrating, but I think SP is simply too sensitive.....

[Eagle Eye]

I do not know ahy you SP would not reproduce result on same day. THere is something wrong here. I think you should be able to reproduce the result atleast from same specimen.

[kim]

I've used Immucor Capture in the past and absolutly hated it!!!! Change methods

[David Saikin]

I have noticed a similar phenomenon in gel. Homozygous jka cells react 1+. However, enzyme pretreatment has removed this reactivity leading me to believe I am seeing complement activity or HLA antibodies that are denatured by the pretreatment. I have seen this in the past with tubes also . . . looks like Jka but enzyme does not enhance.

[yolis76]

I AGREEEEEEE!!!!

Here's another example: I have a patient with a positive screen. Solid phase cell number one (E-e+, K+k+) is neg, cell two (E+e-, K-k+) is 2+. This makes you think Anti-E right? The techs ran the ID panel and the only cells that reacted were 2 out of the three K+ pos cells. The tech then ran the Extend I panel and, again, the only cells that reacted were 2 out of the 4 K+ cells. E was ruled out on 4 cells in solid phase. How can that be? I think were are missing an Anti-E, but how can I prove it when the only cell that reacted was the one cell in the screen? Nothing reacts in the tube, and this patient is pregnant. SOLID PHASE IS A WASTE OF TIME AND MONEY, in my honest opinion.....

[John C. Staley]

I'm curious, any one out there using an antibody detection/identification system that is 100% sensitive, 100% specific and 100% accurate? If so I would sure like to know about it.

:pointandl:faint:

[redwiner]

I agree, nothing is 100%, but at least with Gel we were able to have fairly consistent reactions with 'real' antibodies. Capture is so unpredicatable, and the technical support is poor.

[kate murphy]

I've been using Capture on Galileo for 3 years now, and it is NOT 'poor'. As John has pointed out, nobody's using a system that's 100% specific, sensitive and accurate. Every system has limitations, you have to know your system and your system's limitations.

What we've noticed, and what I'm picking up in this thread, is that OB patients are wierd. Low level SP no-specific reactions - but talking to my colleagues in Boston using Gel - OB patients are also wierd in Gel. How many anti-Lea's did we used to chase in tubes in this same population? The questions is how clinically significant is the reactivity? If it is not reproduceable, if the reactivity is borderline, we do the same as noted earlier - note that it's likely a false pos, and report a neg.

[jgatti]

My institution has been using Capture for close to 10 years. We have been using the Galileo for 3 years. We've been satisfied with the Capture system and the Galileo. I think with any new procedure, there's a learning curve...especially when the new procedure requires subjective interpretation. Performing antibody IDs on an instrument such as the Galileo might improve the consistency of results. We perform a Capture panel when the Capture screen is positive either manually or on the Galileo. We have observed an increased incidence of anti-Jka. Some of these cases were not reproducible using tube testing(we use PeG). None of the patients were OB patients. Since Capture is sensitive, we expect that this may happen. We honor any antibodies ID'd in Capture even if negative with tube testing. We also agree that because of the sensitivity of Capture, it generally cannot be used if the patient has an autoantibody. As far as the patient having a positive JKa typing, I agree that we would need more info (recent TX, DAT results).

[mda]

I am confused with your example. Is the tech trying to rule in Anti-K or Anti-E?

[NedB]

Hello Liz, this is Ned B. You may want to mix fresh serum with your plasma and test with Poly AHG and you may want to do the testing in plastic tubes. Anti-Jka loses its complement reacting component if not stored correctly. We have not used Capture R in quite a while so I don't remember much about the system (whether Capture R uses or can use Poly AHG). We use the gel system exclusively and see no discrepant results - is this good or bad? We hardly ever see something in gel that does not turn out to be a true antibody. My last suggestion would be to try typing the Patient with a different Anti-Jka reagent; Ortho's one does not require AHG.

[marilynm]

I have read all the above comments and examples and wish to say that no one method will detect all antibodies or be perfect every time. I have seen antibodies that react in solid phase that cannot be seen by other methods, the same has been true for gel and also in the tube. How many times have you done something in the tube that was not reproducible, gel too and solid phase?

Specific to anti-Jka, although autoanti-Jka is not common, it does occur.

You must select a method for antibody detection and identification that is best for your lab, learn to deal with the inconsistencies and try to select blood for the patient according to your SOPs.

We use tube routinely in our reference lab but can do gel and soon solid phase and hope to have some data soon on all three methods and compare serological findings with clinical data, which I think is very important.

Marilyn

PS Words of wisdom from the "old" and "wise"?

[OPUS104]

Donor center answer...so beware! We test a pool of 6 donors. Pos. pool = breakdown run individual. (still in SP) Send to ref. (ours) the pos. donor. Gel first...if neg. then PEG....if neg then DAT in Gel and tube. Believe it or not, alot of these are DATs on our donors..(protiens, overcounter drugs?) Anyway, if all neg, release as false pos. Alot of these false pos. have a "funny" pattern or look like a robin egg speckle.

[kharbert]

I work in a Medical Technology program, and don't actually work in the laboratory, but one of our clinical affiliates uses Capture. They noticed when doing manual Capture that the package insert recommends incubation times between 15 minutes and 1 hour. When they started using Capture, they used a 20 minute incubation. The techs noticed that sometimes an antibody would not show a distinct pattern on the 20 minute incubation, but after 45 minutes incubation they could identify the antibody.

They also learned that it is absolutely essential to be sure that the sample is well-centrifuged and that the plasma is truly platelet-poor. Platelets in the plasma give that fuzzy button look.

Kerry

[marilynm]

Dear Kerry: thank you so much for your hints. I know that with every technique there are tips and observations that are good to share with others, whether it be tube, solid phase or gel. I have worked with Capture some and do know that it is important to centrifuge the serum/plasma good before putting it on Capture, and I would guess the same would be with gel.

We will be doing some comparisons soon between tube, gel and solid phase and I will be interested in what antibodies are identifiable at different time frames. I do know that even though commercial antisera says you can incubate 10-15' (whichever the antibody) but that the time can be extended to 30' in some cases and a cell that has weak expression of the antigen might not react until incubation is done for 30'. Fyx (weakened Fyb expression) might be one example, although some examples of anti-Fyb might not react at all, even after 30'.

Sorry for the long reply. I keep forgetting that I said I would try to not be too verbose and I already broke my promise.

Marilyn

[TimOz]

Hi Elvallot,

I suggest that even though we have new technology and automation, the tube LISS additive method is still the "gold standard" method. If that is clean, carry on.

This was the protocol when papain was used as a screening method and many so called "papain only" antibodies were found. The Capture system has advantages and disadvantages but can be viewed the same way.

It is one of the main problems of our science, we have a very tenuios link between serology and clinical relevance and we predict and guess based on peer reviewed published historical cases. Is a "Capture only" antibody clinically relevant? You don't know and you can't know until someone looks at clinical cases but I can't see that happening.

[RonL.]

Hi,

Maybe the positive Jka typing was really the baby's phenotype, since she was an ob patient. Solid phase is pretty sensitive. Possibly mom's ab's are now sitting on the baby's cells, accounting for her negative post screen. You can retest the neg screen in solid phase, incubating longer, up to an hour, to see if anything shows up.

It would be interesting to see if the baby has a positive direct coombs upon birth.

[John Eggington]

There is something (or a range of ‘somethings’) out there that is/are mimicking anti-Jka. I saw it a lot, 15-20 years ago (in gel, mostly, by enzyme technique), but it seemed to have stopped happening. The past year or so seems to see it happening again, but now we’re finding it with a, gel, enzyme IAT technique (although our workload has risen by a great deal, so it could be a ‘numbers’ issue). So, if the patient happens to be Jk(a+), you’ll call it ‘auto’, if your patient happens to be Jk(a-) you’ll call it ‘allo’, but actually it isn’t either. This thread shows the phenomenon happens with other techniques. Maybe one of these techniques is better at detecting these ‘non-anti-Jka’, and so looks as if it’s more sensitive for this specificity? Whatever it is, it’s a nuisance!

[Malcolm Needs ☆]

I'm not saying you are incorrect John, but you would have to prove that by adsorbing the "non-anti-Jka" to exstinction with Jk(a-b+) red cells.

[John Eggington]

Maybe my choice of the term ‘mimicking’ wasn’t so good. I wasn’t thinking of the classic mimic, which could be adsorbed ‘out’ by cells lacking the relevant antigen. More something, or things, that behaved as ‘anti-Jka’, when the conditions were right (e.g., the unnatural ones found in the various techniques we use to detect antibodies). That’s not to say they wouldn’t be adsorbed ‘out’ by Jk(a-) cells, just that a failure to adsorb wouldn’t rule out the possibility of it not being a ‘real’ anti-Jka. Hmmm...I think I know what I mean, anyway!?